Methods for assaying cellular binding interactions

ABSTRACT

There are provided methods, and devices for assaying for a binding interaction between a protein, such as a monoclonal antibody, produced by a cell, and a biomolecule. The method may include retaining the cell within a chamber having an aperture; exposing the protein produced by the cell to a capture substrate, wherein the capture substrate is in fluid communication with the protein produced by the cell and wherein the capture substrate is operable to bind the protein produced by the cell; flowing a fluid volume comprising the biomolecule through the chamber via said aperture, wherein the fluid volume is in fluid communication with the capture substrate; and determining a binding interaction between the protein produced by the cell and the biomolecule.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/365,237 entitled “METHODS FOR ASSAYING CELLULARBINDING INTERACTIONS” filed on 16 Jul. 2010, which is incorporatedherein by reference in its entirety.

FIELD OF INVENTION

This invention relates to the field of microfluidics and proteinbinding, more specifically, binding interaction between biomolecules.

BACKGROUND

Antibodies are defense proteins produced by the vertebrate adaptiveimmune system for the purposes of binding and targeting for clearance ofa diverse range of bacteria, viruses, and other foreign molecules(collectively referred to as antigens) (see, for e.g., Abbas et al.(1997), Cellular and Molecular Immunology, 3^(rd) Ed., Chapter 3, pp.37-65). As a result of their ability to bind target antigens selectivelyand with high affinity, antibodies are useful tools for proteinpurification, cell sorting, diagnostics, and therapeutics.

Conventional antibody production has involved the immunization ofanimals (i.e., mice) with a target antigen, such as a virus, bacteria,foreign protein, or other molecule. The immunized mice produce on theorder of 10⁴-10⁵ antibody secreting cells (ASCs), each with the capacityto produce a unique (monoclonal) antibody specific to the target antigen(see, for e.g., Poulson et al. (1997), J. Immunol. 179: 3841-3850; andBabcock et al. (1996), Proc. Natl. Acad. Sci. USA 93: 7843-7848).

The ASCs are then harvested from the immunized animals and screened inorder to select which cells are producing antibodies of desired affinityand selectivity to the target antigen. Since single ASCs do not produceantibodies in sufficiently large quantities for binding affinitymeasurements, each ASC is clonally expanded. Primary ASCs do not growefficiently in laboratory tissue cultures; thus, clonal expansion may beachieved by fusing ASCs to murine myeloma (cancer) cells to produceimmortalized, antibody-secreting (hybridoma) cells (see, for e.g.,Kohler, G. and Milstein, C. (1975), Nature 256: 495-497). Using thismethod, expansion of each successfully created hybridoma then produces amonoclonal antibody in sufficiently high concentrations to measure itsaffinity and selectivity to a target antigen.

It has been recognized that a limitation of hybridoma technology is thelow efficiency of the fusion process. For example, whereas an immuneresponse may produce on the order of 10⁴-10⁵ antibody secreting cells, atypical fusion will yield less than 100 viable hybridomas. (see, fore.g., Kohler, G. and Milstein, C. (1975), Nature 256: 495-497; Karpas etal. (2001), Proc. Natl. Acad. Sci. USA 98: 1799-1804; and Spieker-Poletet al. (1995), Proc. Natl. Acad. Sci. USA 92: 9348-9352). Therefore,fusions from hundreds to thousands of animals are required to fullysample the diversity of antibodies produced in an immune response,making the hybridoma approach both time-consuming and expensive.Attempts to circumvent hybridoma generation by immortalizingantibody-producing cells using viral transformations have resulted inmodest gains in the efficiency of ASC immortalization. However, theseapproaches still require costly and time-consuming clonal expansion inorder to produce sufficient quantities of monoclonal antibodies toscreen for affinity and selectivity to target antigens (see for e.g.,Pasqualini, R. and Arap, W. (2004), Proc. Natl. Acad. Sci. USA 101:257-259; Lanzavecchia et al. (2007), Current Opinion in Biotechnology18: 523-528; and Traggiai et al. (2004), Nat Med 10: 871-875).

Devices have been developed to estimate the equilibrium dissociationconstants of antibodies secreted from single antibody-secreting cells(Story, C. M. et al. Proc. Natl. Acad. Sci. U.S.A. (2008)105(46):17902-17907; and Jin, A. et al. Nat. Med. (2009)15(9):1088-1092), but do not measure antibody-antigen binding kineticsusing antibodies secreted from single cells.

SUMMARY

In a first embodiment, there is provided a method of assaying for abinding interaction between a protein produced by a cell and abiomolecule: (a) retaining the cell within a chamber having an inlet andan outlet; (b) exposing the protein produced by the cell to a capturesubstrate, wherein the capture substrate is in fluid communication withthe protein produced by the cell and wherein the capture substrate isoperable to bind the protein produced by the cell; (c) flowing a firstfluid volume comprising the biomolecule through the inlet into thechamber and out the outlet, wherein the first fluid volume is in fluidcommunication with the capture substrate; and (d) determining bindinginteractions between the protein produced by a cell and the biomolecule.

The cell may be an antibody producing cell (APC), the protein producedby the cell is an antibody and the biomolecule is an antigen. The cellmay be a single cell. The biomolecule may be a fluorescently labeledantigen. The determining binding interactions may be a measure ofantigen-antibody binding kinetics. The determining the antigen-antibodybinding kinetics may include fluorescence imaging of antigen-antibodybinding. The determining the binding interactions may be by one or moreof the following techniques: surface plasmon resonance (SPR)spectroscopy, fluorescence anisotropy, interferometry, or fluorescenceresonance energy transfer (FRET). The determining of the bindinginteraction may be by a nanocalorimeter or a nanowire nanosensor. Themeasure of antigen-antibody binding kinetics may be the K_(on) rate. Themeasure of antigen-antibody binding kinetics may be the K_(off) rate.The measure of antigen-antibody binding kinetics may be the both theK_(on) rate and the K_(off) rate. The protein produced by the cell maybe an antibody. The antibody may be a monoclonal antibody. The proteinproduced by the cell may be an antigen. The biomolecule may be anantigen. The biomolecule may be selected from one of the following: anantibody, a whole cell, a cell fragment, a bacterium, a virus, a viralfragment, and a protein. The protein produced by the cell may not besecreted by the cell, and the method may further include a step of celllysis prior to exposing the protein produced by the cell to the capturesubstrate. The protein produced by the cell may not be secreted by thecell, and the method may further include a step of cell lysis afterexposing the protein produced by the cell to the capture substrate. Thecapture substrate may be a removable capture substrate. The removablecapture substrate may be an anti-Ig bead. The removable capturesubstrate may be an anti-Ig bead and/or oligo (dT) bead. The removablecapture substrate may include a capture substrate capable of capturingboth nucleic acids and antibodies. The removable capture substrate mayinclude a capture substrate capable of capturing nucleic acids and acapture substrate capable of capturing antibodies. The removable capturesubstrate may include a capture substrate capable of capturing nucleicacids. The binding of the antibodies may be further tested by viralinactivation. The binding of the antibodies may be further tested bybacterial inactivation. The binding of the antibodies may be furthertested by cell inactivation. The method may further include adding thecell to a reverse transcription polymerase chain reaction (RT-PCR)reaction to amplify the heavy and light chain genes. The amplificationmay be performed in a number of ways. For example, 1) the cells may beeluted into RT-PCR mix containing primers for both heavy and light chaingenes for multiplex amplification of both genes in a single reaction.Alternatively, the cells may be eluted into RT-PCR mix without primers,the mix may then be split into two equal volume aliquots and therespective heavy and light chain primers may be added to the twoaliquots for single-plex amplification. Both methods have been shown towork to amplify the heavy and light chains from a single cell. Theexposing the protein produced by the cell to the capture substrate mayinclude flowing a removable capture substrate into the chamber. Themethod may further include washing the cell prior to flowing a removablecapture substrate into the chamber. The protein produced by the cell maybe an antigen and the biomolecule may be an antibody. The antibody maybe a monoclonal antibody. The biomolecule may be a fluorescently labeledantibody. The fluorescently labeled antibody may be a monoclonalantibody. The determining binding interactions may be a measure ofantigen-antibody binding kinetics. The measure of antigen-antibodybinding kinetics may be any one or both of: a K_(on) rate; and a K_(off)rate. The APC may be from one of the following: a human, a rabbit, arat, a mouse, a sheep, an ape, a monkey, a goat; a dog, a cat, a camel,or a pig. The removable capture substrate may be a carboxylic acid(COOH) functionalized bead. The removable capture substrate may becapable of binding the protein produced by the cell and the nucleicacids encoding the protein produced by the cell. The method may furtherinclude washing the cell prior to exposing the protein produced by thecell to a capture substrate. The APC may be selected from one of thefollowing: a primary B cell and a memory B cell.

In a further embodiment, there is provided a cell assay method, themethod including: distributing an antibody producing cell (APC) to achamber, wherein the APC is in a first fluid; replacing the first fluidwith a second fluid while maintaining the APC in the chamber; placingthe antibodies produced by the APC in fluid communication with anantigen; and determining the antigen-antibody binding kinetics of theantibodies produced by the APC with the antigen.

In a further embodiment, there is provided a method of assaying for abinding interaction between a protein produced by a cell and abiomolecule, the method including: (a) retaining the cell within achamber having an aperture; (b) exposing the protein produced by thecell to a capture substrate, wherein the capture substrate is in fluidcommunication with the protein produced by the cell and wherein thecapture substrate is operable to bind the protein produced by the cell;(c) flowing a fluid volume comprising the biomolecule through thechamber via said aperture, wherein the fluid volume is in fluidcommunication with the capture substrate; and (d) determining a bindinginteraction between the protein produced by the cell and thebiomolecule.

The measure of antigen-antibody binding kinetics may be the K_(on) rate.The measure of antigen-antibody binding kinetics may be the K_(off)rate. The measure of antigen-antibody binding kinetics may be the boththe K_(on) rate and the K_(off) rate. The binding of the antibodies maybe further tested by viral inactivation. The binding of the antibodiesmay be further tested by bacterial inactivation. The binding of theantibodies may be further tested by cell inactivation. The determiningof antigen-antibody binding kinetics may be by one or more of thefollowing techniques: surface plasmon resonance (SPR) spectroscopy,fluorescence anisotropy, interferometry, or fluorescence resonanceenergy transfer (FRET). The determining of antigen-antibody bindingkinetics may be by a nanocalorimeter or a nanowire nanosensor. Themethod may further include adding the cell to a reverse transcriptionpolymerase chain reaction (RT-PCR) reaction to amplify the heavy andlight chain genes. The placing the antibodies produced by the APC influid communication with an antigen may include flowing a removablecapture substrate into the chamber. The method may further includewashing the cell prior to flowing a removable capture substrate into thechamber. The APC may be from one of the following: a human, a rabbit, arat, a mouse, a sheep, an ape, a monkey, a goat; a dog, a cat, a camel,or a pig. The removable capture substrate may be a carboxylic acid(COOH) functionalized bead. The removable capture substrate may becapable of binding the protein produced by the cell and the nucleicacids encoding the protein produced by the cell. The method may furtherinclude washing the cell prior to exposing the protein produced by thecell to a capture substrate. The APC may be selected from one of thefollowing: a primary B cell and a memory B cell. The method may furtherinclude adding a removable capture substrate to the chamber to capturethe antibodies produced by the APC prior to placing the antibodiesproduced by the APC in fluid communication with an antigen. The placingof the antibodies produced by the APC in fluid communication with anantigen may include flowing a fluorescently labeled antigen through thechamber. The method may further include collecting the mRNA from thecell for a reverse transcription polymerase chain reaction (RT-PCR)reaction to amplify the heavy and light chain genes. The determining theantigen-antibody binding kinetics may include fluorescence imaging ofantigen-antibody binding.

In a further embodiment, there is provided a microfluidic device forassaying for a binding interaction between a protein produced by a celland a biomolecule, the device comprising: a chamber, having: (i) atleast one inlet; (ii) at least one outlet; and (iii) a reversible traphaving spaced apart structural members extending across the chamber toseparate the at least one inlet and at least one outlet wherein thespaced apart structural members are operable to allow fluid flow throughthe chamber from the inlet to the outlet while providing size selectionfor a particle within the fluid flow.

In a further embodiment, there is provided a microfluidic device forassaying for a binding interaction between a protein produced by a celland a biomolecule, the device comprising: a chamber, having: (i) atleast one inlet; (ii) at least one outlet; and (iii) a reversible trap,wherein the reversible trap is a narrowing of the chamber from to allowfluid flow through the chamber from the inlet to the outlet whileproviding size selection for a particle within the fluid flow.

In a further embodiment, there is provided a microfluidic device forassaying a binding interaction between a protein produced by a cell anda biomolecule, the device including: a chamber having an aperture and achannel for receiving a flowed fluid volume through the chamber via saidaperture, the channel providing size selection for a particle withinsaid fluid volume.

In a further embodiment, there is provided a microfluidic device forassaying a binding interaction between a protein produced by a cell anda biomolecule, the device including: a chamber having an aperture; areversible trap having spaced apart structural members extending acrossthe chamber, the structural members being operable to allow a fluidvolume to flow through the chamber while providing size selection for aparticle within said fluid volume.

The distance between the spaced apart structural members may be lessthan or equal to about 4.6 microns. The distance between the spacedapart structural members may be less than or equal to about 4.5 microns.The distance between the spaced apart structural members may be lessthan or equal to about 4.4 microns. The distance between the spacedapart structural members may be less than or equal to about 4.3 microns.The distance between the spaced apart structural members may be lessthan or equal to about 4.2 microns. The distance between the spacedapart structural members may be less than or equal to about 4.1 microns.The distance between the spaced apart structural members may be lessthan or equal to about 4.0 microns. The distance between the spacedapart structural members may be less than or equal to about 3.9 microns.The distance between the spaced apart structural members may be lessthan or equal to about 3.8 microns. The distance between the spacedapart structural members may be less than or equal to about 3.7 microns.The distance between the spaced apart structural members may be lessthan or equal to about 3.6 microns. The distance between the spacedapart structural members may be less than or equal to about 3.5 microns.The distance between the spaced apart structural members may be lessthan or equal to about 3.4 microns. The distance between the spacedapart structural members may be less than or equal to about 3.3 microns.The distance between the spaced apart structural members may be lessthan or equal to about 3.2 microns. The distance between the spacedapart structural members may be less than or equal to about 3.1 microns.The distance between the spaced apart structural members may be lessthan or equal to about 3.0 microns. The distance between the spacedapart structural members may be less than or equal to about 2.9 microns.The distance between the spaced apart structural members may be lessthan or equal to about 2.8 microns. The distance between the spacedapart structural members may be less than or equal to about 2.7 microns.The distance between the spaced apart structural members may be lessthan or equal to about 2.6 microns. The distance between the spacedapart structural members may be less than or equal to about 2.5 microns.The distance between the spaced apart structural members may be lessthan or equal to about 2.4 microns. The distance between the spacedapart structural members may be less than or equal to about 2.3 microns.The distance between the spaced apart structural members may be lessthan or equal to about 2.2 microns. The distance between the spacedapart structural members may be less than or equal to about 2.1 microns.The distance between the spaced apart structural members may be lessthan or equal to about 2.0 microns. The distance between the spacedapart structural members may be less than or equal to about 1.9 microns.The distance between the spaced apart structural members may be lessthan or equal to about 1.8 microns. The distance between the spacedapart structural members may be less than or equal to about 1.7 microns.The distance between the spaced apart structural members may be lessthan or equal to about 1.6 microns. The distance between the spacedapart structural members may be less than or equal to about 1.5 microns.The distance between the spaced apart structural members may be lessthan or equal to about 1.4 microns. The distance between the spacedapart structural members may be less than or equal to about 1.3 microns.The distance between the spaced apart structural members may be lessthan or equal to about 1.2 microns. The distance between the spacedapart structural members may be less than or equal to about 1.1 microns.The distance between the spaced apart structural members may be lessthan or equal to about 1.0 microns. The distance between the spacedapart structural members may be less than or equal to about 0.9 microns.The distance between the spaced apart structural members may be lessthan or equal to about 0.8 microns. The distance between the spacedapart structural members may be less than or equal to about 0.7 microns.The distance between the spaced apart structural members may be lessthan or equal to about 0.6 microns. The distance between the spacedapart structural members may be less than or equal to about 0.5 microns.The spaced apart structural members may be posts. The spaced apartstructural members may be between 5 to 30 microns in width. The spacedapart structural members may be between 10 to 20 microns in width. Thespaced apart structural members may be between 5 to 30 microns in width.The spaced apart structural members may be between 5 to 20 microns inwidth. The spaced apart structural members may be between 5 to 10microns in width.

The narrowing of the chamber may be from greater than about 10 micronsto less than about 5.0 microns. The narrowing of the chamber may be fromgreater than about 10 microns to less than about 4.9 microns. Thenarrowing of the chamber may be from greater than about 10 microns toless than about 4.8 microns. The narrowing of the chamber may be fromgreater than about 10 microns to less than about 4.7 microns. Thenarrowing of the chamber may be from greater than about 10 microns toless than about 4.6 microns. The narrowing of the chamber may be fromgreater than about 10 microns to less than about 4.5 microns. Thenarrowing of the chamber may be from greater than about 10 microns toless than about 4.4 microns. The narrowing of the chamber may be fromgreater than about 10 microns to less than about 4.3 microns. Thenarrowing of the chamber may be from greater than about 10 microns toless than about 4.2 microns. The narrowing of the chamber may be fromgreater than about 10 microns to less than about 4.1 microns. Thenarrowing of the chamber may be from greater than about 10 microns toless than about 4.0 microns.

It will be appreciated by a person of skill in the art that the distancebetween the spaced apart structural members and the narrowing of thechamber to produce the reversible trap, will depend on the size of thecells being assayed and the size of the removable capture substrate, andthe flow velocity through the chamber, whereby the cell and theremovable capture substrate are retained in the chamber at a first flowvelocity and whereby the removable capture substrate is retained in thechamber and the cell is able to deform and fit through the reversibletrap at a second flow velocity. Alternatively, there may be differentsized removable capture substrates and some may be permitted to passthrough the reversible trap, while other may be retained. Furthermore,there may be further flow velocities possible with a given device,whereby the reversible trap may deform to allow the removable capturesubstrates to pass through the chamber. Alternatively, the chamber maybe pierced to remove the removable substrate and/or cells. The narrowingof the chamber may correspond to the channel size selection.

The particle may be selected from one or more of the cell, thebiomolecule, the protein, the protein bound to a removeable capturesubstrate, and the removeable capture substrate. The size selection ofthe reversible trap may prevent the cell and the removeable capturesubstrate from passing through the reversible trap, and may allow thebiomolecule and the protein to pass through the reversible trap at afirst flow velocity, and the size selection of the reversible trap mayprevent the removeable capture substrate from passing through thereversible trap, while allowing the cell, the biomolecule and theprotein to pass through the reversible trap at a second flow velocity.The outlet may be a sieve valve and the flow velocity through thechamber when the valve is in an open position may be sufficient to allowthe cell to deform and pass through the reversible trap. The device maybe operable to provide two or more flow velocities through the chamber.The device may be operable to provide two flow velocities through thechamber. The device may be operable to provide three flow velocitiesthrough the chamber. The device may be operable to provide four flowvelocities through the chamber. The microfluidic device may be operableto allow for removal of the removeable capture substrate. Themicrofluidic device may be operable to allow for removal of the cell.

The cells get trapped in the chambers when the sieve valves are closed.However, as with the posts, the cells deform when the sieve valve isopened and there is increased flow through the chambers. Bothimplementations of the reversible trap have worked, but the bead postdesign is slightly more robust at retaining the beads. The cells beingused in the present experiments are about 10 microns in diameter, thebeads are 5 microns in diameter, and the space between the posts is lessthan 3 microns.

A chamber may be in fluid communication with a first auxiliary chamber,wherein there is may be a valve between the chamber and the firstauxiliary chamber. The first auxiliary chamber may be in fluidcommunication with a second auxiliary chamber, wherein there is a valvebetween the first and second auxiliary chambers, wherein the valve hasan open position to allow fluid flow from the first auxiliary chamber tothe second auxiliary chamber and a closed position to prevent fluid flowfrom the first auxiliary chamber to the second auxiliary chamber. Thefirst auxiliary chamber may be in fluid communication with a secondauxiliary chamber and the second auxiliary chamber is in fluidcommunication with a third auxiliary chamber, wherein there is a valvebetween the first and second auxiliary chambers, wherein the valve hasan open position to allow fluid flow from the first auxiliary chamber tothe second auxiliary chamber and a closed position to prevent fluid flowfrom the first auxiliary chamber to the second auxiliary chamber,wherein there is a valve between the second and third auxiliarychambers, wherein the valve has an open position to allow fluid flowfrom the second auxiliary chamber to the third auxiliary chamber and aclosed position to prevent fluid flow from the second auxiliary chamberto the third auxiliary chamber. The volumes of the first second andthird auxiliary chambers relative to the chamber may be such that fluidmay be flowed into these chambers such that subsequent RT and PCR orother reactions may be carried out without exchanging the fluid (forexample, where a first outlet is in a closed position).

The volume of the auxiliary chambers may be expandable. The volume ofthe chamber may be between 0.1 nL to 100.0 nL. The unexpanded volume ofthe expandable the chamber may be between 0.1 nL to 100.0 nL. The volumeof the chamber may be 0.6 nL. The unexpanded chamber may be 0.6 nL. Theeffective volume of a given chamber may be increased by expanding theinitial chamber or by opening a valve to provide fluid flow into one ormore auxiliary chambers. The ratio between the second auxiliary chamberand the first auxiliary chamber may be 5:1. The ratio between the secondauxiliary chamber and the first auxiliary chamber may be at least 5:1.The ratio between the expanded chamber and the unexpanded chamber may be5:1 or the ratio between the expanded first auxiliary chamber unexpandedfirst auxiliary chamber may be 5:1. The ratio between the expandedchamber and the unexpanded chamber may be at least 5:1 or the ratiobetween the expanded first auxiliary chamber unexpanded first auxiliarychamber may be at least 5:1. The ratio between the second auxiliarychamber and the first auxiliary chamber, or between the expanded chamberand the unexpanded chamber, or between the expanded first auxiliarychamber unexpanded first auxiliary chamber may vary depending on thereaction mixtures chosen, the concentrations of the components of themixture and the concentration of the material being assayed.Alternatively, the chamber may be between 0.05 nL and 100.0 nL.Alternatively, the chamber may be between 0.05 nL and 90.0 nL.Alternatively, the chamber may be between 0.1 nL and 95.0 nL.Alternatively, the chamber may be between 0.1 nL and 90.0 nL.Alternatively, the chamber may be between 0.1 nL and 85.0 nL.Alternatively, the chamber may be between 0.1 nL and 80.0 nL.Alternatively, the chamber may be between 0.1 nL and 75.0 nL.Alternatively, the chamber may be between 0.1 nL and 70.0 nL.Alternatively, the chamber may be between 0.1 nL and 65.0 nL.Alternatively, the chamber may be between 0.1 nL and 60.0 nL.Alternatively, the chamber may be between 0.1 nL and 55.0 nL.Alternatively, the chamber may be between 0.1 nL and 50.0 nL.Alternatively, the chamber may be between 0.1 nL and 45.0 nL.Alternatively, the chamber may be between 0.1 nL and 40.0 nL.Alternatively, the chamber may be between 0.1 nL and 35.0 nL.Alternatively, the chamber may be between 0.1 nL and 30.0 nL.Alternatively, the chamber may be between 0.1 nL and 25.0 nL.Alternatively, the chamber may be between 0.1 nL and 20.0 nL.Alternatively, the chamber may be between 0.1 nL and 15.0 nL.Alternatively, the chamber may be between 0.1 nL and 10.0 nL.Alternatively, the chamber may be between 0.1 nL and 9.0 nL.Alternatively, the chamber may be between 0.1 nL and 8.0 nL.Alternatively, the chamber may be between 0.1 nL and 7.0 nL.Alternatively, the chamber may be between 0.1 nL and 6.0 nL.Alternatively, the chamber may be between 0.1 nL and 5.0 nL.Alternatively, the chamber may be between 0.1 nL and 4.0 nL.Alternatively, the chamber may be between 0.1 nL and 3.0 nL.Alternatively, the chamber may be between 0.1 nL and 2.0 nL.Alternatively, the chamber may be between 0.1 nL and 1.0 nL.

In a further embodiment, there is provided a method of assaying for aprotein of interest produced by a cell, the method comprising:incubating the cell with a removable capture substrate in a buffer,wherein the removable capture substrate is capable of binding theprotein of interest and nucleic acids encoding the protein of interest;and screening the bound removable capture substrate to determine whetherthe cell produces the protein of interest.

In a further embodiment, there is provided a method of assaying for aprotein of interest produced by a cell, the method comprising:incubating the cell with a removable capture substrate in a buffer,wherein the removable capture substrate is capable of binding theprotein of interest; and screening the bound removable capture substrateto determine whether the cell produces the protein of interest.

In a further embodiment, there is provided a method of identifying amonoclonal antibody of interest, the method comprising: incubating anAPC with a removable capture substrate in a suitable buffer, wherein theremovable capture substrate is capable of binding the monoclonalantibody produced by the APC and nucleic acids encoding the variableregions of the monoclonal antibody; and screening the bound removablecapture substrate to determine whether the APC produces the monoclonalantibody of interest.

In a further embodiment, there is provided a cell assay method, themethod comprising: distributing an APC to a chamber, wherein there is onaverage one APC in the chamber, wherein the APC is incubated with aremovable capture substrate in a first solution, and wherein theremovable capture substrate is capable of binding an antibody ofinterest produced by the APC and nucleic acids encoding the variableregions of the antibody of interest; replacing the first solution with asecond solution while maintaining the APC in the chamber; placing theantibody of interest produced by the APC in fluid communication with anantigen; and screening the bound removable capture substrate todetermine whether the APC produces the antibody of interest.

In a further embodiment, there is provided a method of assaying for achemical interaction between a protein produced by a cell and abiomolecule, the method comprising: distributing the cell to a chamber,wherein the cell is in a first solution; replacing the first solutionwith a second solution while maintaining the cell in the chamber;placing the protein in fluid communication with the biomolecule; andtesting the chemical interaction of the protein produced by the cellwith the biomolecule.

In a further embodiment, there is provided a method of identifying amonoclonal antibody of interest, the method comprising: incubating anantibody producing cell (APC) with a removable capture substrate in asuitable buffer, wherein the removable capture substrate is capable ofbinding the monoclonal antibody produced by the APC and nucleic acidsencoding the variable regions of the monoclonal antibody; and screeningthe bound removable capture substrate to determine whether the APCproduces the monoclonal antibody of interest.

In a further embodiment, there is provided a cell assay method, themethod comprising: distributing an antibody producing cell (APC) to achamber, wherein there is on average one APC in the chamber, wherein theAPC is incubated with a removable capture substrate in a first solution,and wherein the removable capture substrate is capable of binding anantibody of interest produced by the APC and nucleic acids encoding thevariable regions of the antibody of interest; replacing the firstsolution with a second solution while maintaining the APC in thechamber; placing the antibody of interest produced by the APC in fluidcommunication with an antigen; and screening the bound removable capturesubstrate to determine whether the APC produces the antibody ofinterest.

In a further embodiment, there is provided a method of assaying for aprotein of interest produced by a cell. The method involves incubatingthe cell with a removable capture substrate in a suitable buffer,wherein the removable capture substrate is capable of binding theprotein of interest; and screening the bound removable capture substrateto determine whether the cell produces the protein of interest.

The method may involve determining the binding affinity of the proteinof interest. The method may involve determining a dissociation rate; andassociation rate and dissociation rate. The method may involve lysingthe cell prior to incubation with the removable capture substrate,wherein the protein of interest is not secreted by the cell.

In a further embodiment, there is provided an device for selecting acell that produces a protein having a binding affinity for abiomolecule. The device may include a microfluidic device as describedherein operably configured to hold an aliquot, wherein the aliquot onaverage contains one cell, and wherein the protein produced by the cellis in fluid communication with the biomolecule; and a detector fordetecting the binding affinity of the protein produced by the cell.

The device may include a detector that is a fluorescence imager fordetecting the binding affinity. The device may include a detector thatis a surface plasmon resonance (SPR) spectroscopy apparatus, or afluorescence anisotropy apparatus, or an interferometry apparatus, or aFRET apparatus. Further, the device may include a detector that is ananocalorimeter or a nanowire nanosensor.

In a further embodiment, there is provided a kit for identifying a cellthat produces antibodies having a binding affinity for an antigen. Thekit includes a microfluidic device as contemplated herein; and aremovable capture substrate. The kit may include the removable capturesubstrate being capable of binding proteins, or nucleic acids, orproteins and nucleic acids. The kit may include the removable capturesubstrate being a microsphere. Further, the kit may include themicrosphere being a polystyrene bead or a silica bead. Further, the kitmay include the microsphere being a carboxylic acid (COOH)functionalized bead.

The kit may include an antigen label. The kit may include an antigenlabel that is a fluorescent label. Further, the kit may includeinstructions for the use of the device contemplated herein to identify acell that produces proteins having a desired binding affinity. Further,the kit may include instructions for immunizing an animal and collectingAPCs. Further, the kit may include an antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a microfluidic device and schematics for bead-basedmeasurements of antibody-antigen binding kinetics. Panel (A) is anillustration of a microfluidic device containing control channels forindividually selecting six reagent inlets and actuating sieve valves onthe reagent outlet channel. Panel (B) shows a microscopic image of thedevice with food coloring to visualize distinct reagent inlets (asshown) and control channels (as shown) (5× magnification); Top insetdepicts a close-up of beads trapped using sieve valves (20×magnification; Bottom inset depicts fluorescence image of beads duringbinding kinetic measurements (100× magnification). Panel (C) shows aschematic of a bead assay for direct measurement of association anddissociation kinetics of immobilized mAbs and fluorescently-labeledantigen. Panel (D) shows a variation of a bead assay for indirectmeasurement of dissociation kinetics of immobilized mAbs and unlabeledantigen molecules.

FIG. 2 shows a schematic diagram of an embodiment of a microfluidicdevice for the detection of antibody secreted from single cells. (A)Hydraulic pressure is applied to valves (fully-closing) and sieve valves(partially-closing) formed by the intersection of actuation controlchannels with rounded- or square-profile flow channels, respectively.(B) An expanded view of an embodiment of a microfluidic device for thedetection of antibody secreted from single cells. (1) chip is flushedwith 1×PBS; (2) antibody-secreting cells and antibody-capture beads areloaded into chambers; (3) cells are incubated for one hour to allow forantibody secretion; (4): mix valve is opened to allow for secretedantibody to bind to beads; (5) beads and cells are captured againstsieve valve and unbound antibody is washed out; (6) chambers flushedwith fluorescently-labeled antigen; image and measure antibody-antigenassociation kinetics; and (7) flushed out unbound antigen with 1×PBS;image and measure antibody-antigen dissociation kinetics.

FIG. 3 shows plots of microfluidic bead-based measurements ofantibody-antigen binding kinetics. Direct fluorescent measurements ofassociation and dissociation kinetics of (A) D1.3 mAb and HEL-Dylight488conjugate, (B) HyHEL-5 mAb and HEL-Dylight488 conjugate, (C) LGB-1 mAband enhanced green fluorescent protein (EGFP) are demonstrated. (D)Indirect measurement of dissociation kinetics of D1.3 mAb and HEL usingHEL-Dylight488 conjugate is demonstrated.

FIG. 4 shows simultaneous measurement of multiple antibody-antigenbinding kinetics using optical and spatial multiplexing. (A) Plotsmeasured association and dissociation kinetics of 3 distinct mAbs(HyHEL-5, D1.3, and LGB-1 mAb) interacting with 2 different antigens(HEL-Dylight633 conjugate and EGFP) is demonstrated. (B) A micrographshowing false-coloured, overlay of images taken with distinctfluorescence filter cubes to identify anti-lysozyme mAbs and anti-EGFPmAbs is demonstrated.

FIG. 5 shows plots of sensitivity and detection limit ofantibody-antigen binding kinetics measurements. (A) Measured associationkinetics of D1.3 mAb-Dylight488 conjugate on rabbit anti-mouse pAbcoated beads is demonstrated. Inset demonstrates a schematic of beadassay for measuring binding kinetics of fluorescently-labeled mouse mAband rabbit anti-mouse pAb coated beads. (B) Association kinetics ofHEL-Dylight488 conjugate on beads with varying amounts of immobilizedD1.3 mAb is demonstrated. (C) Equilibrium bead fluorescence varieslinearly with the amount of immobilized D1.3 mAb. Inset shows a close-upof the graph to highlight detection limit of 1% bead coverage. (D)Direct measurement of equilibrium dissociation constants by measuringequilibrium bead fluorescence using immobilized D1.3 mAb and varyingconcentrations of HEL-Dylight488.

FIG. 6 shows antibody-antigen binding kinetics measured using antibodiessecreted from a single cell. (A) Microscope image of D1.3 hybridoma cellloaded into a microfluidic device adjacent to rabbit anti-mouse pAbcoated beads trapped using a sieve valve is shown. (B) “Single-cycle”binding kinetics from a single bead containing D1.3 mAbs secreted from asingle cell and subject to increasing concentrations of HEL-Dylight488conjugate is demonstrated.

FIG. 7 shows the effect of fluorophore stability on measuredantibody-antigen binding kinetics. (A) Photobleaching rates offluorescent dye molecules under 100 W Hg lamp illumination using 100×oil-immersion objective (NA 1.30) are plotted. (B) Effect of fluorescentexposure times on measured association kinetics of D1.3 mAb andHEL-Dylight488 are plotted.

FIG. 8 shows the effect of different bead immobilization chemistries onmeasured antibody-antigen binding kinetics. Measured kinetics areunaffected by bead composition (silica or polystyrene) or by differentpolyclonal capture antibodies (rabbit or goat pAbs).

FIG. 9 shows a plot of measured dissociation kinetics of mouse mAb fromantibody capture beads. No dissociation of D1.3 mAb-Dylight488 conjugatefrom Rabbit anti-Ms pAb coated beads was observed over 3 days.

FIG. 10 shows a plot of the effect of antigen re-binding on measuredantibody-antigen dissociation kinetics. Dissociation kinetics of D1.3mAb and HEL-Dylight488 conjugate were unaffected by the presence of alarge concentration of competitive antigen (2 mg/mL HEL).

FIG. 11 shows a plot of the effect of mass transport on measuredantibody-antigen binding kinetics. Association and dissociation kineticsof D1.3 mAb and HEL-Dylight488 conjugate were unaffected by varying flowrates over a range of ˜3-15 μL/hr.

FIG. 12 shows representative microscopic images of primary ASCs in amicrofluidic chamber in fluid communication with antibody capture beadsand oligo(dT) beads.

FIG. 13 shows an image of an ELISPOT control assay confirming that thecells depicted in FIG. 12 are ASCs. The left image represents cells thatsecreted any antibody; the right image represents only those cells thatsecreted HEL-specific antibodies.

FIG. 14 shows a scheme for preparing dual-capture (i.e., dual-function)beads using carbodiimide chemistry.

FIG. 15 shows images of dual-function beads. Polystyrene COOH beads wereconjugated with rabbit anti-mouse pAb and amine functionalizedoligo(dT)₂₅ using carbodiimide chemistry. (A) Brightfield image ofdual-function beads trapped using microfluidic sieve valve. (B)Fluorescence image of synthetic single-stranded DNA molecules capturedon dual-function beads. Synthetic DNA molecules are labeled with Cy5fluorophore for visualization and also contain a poly(A) tail that bindsto the oligo(dT) on the bead surface. (C) Fluorescence image of mouseD1.3 monoclonal antibody (mAb) captured on dual-function beads. D1.3mAbs are labeled with Dylight488 fluorophore for visualization and bindto the Rabbit anti-Mouse pAb on the bead surface.

FIG. 16 shows a microscopic image (A) and antibody-antigen bindingkinetics (B) as determined from a microfluidic device for dual purposebeads.

FIG. 17 depicts (A) K_(d), (B) K_(on), and (C) K_(off) rates determinedfrom specific eluted chambers according to Example 9 herein.

FIG. 18 shows representative fluorescence intensity data over time forspecific eluted chambers according to Example 9 herein. (A) depicts datafor R00C04; (B) depicts data for R04C06.

FIG. 19 shows Kappa chain results from the first round of RT-PCR areshown in FIG. 19, Panel A. Kappa chain results from the second round ofRT-PCR are shown in FIG. 19, Panel B. Heavy chain results from the firstround of RT-PCR are shown in FIG. 19, Panel C. Heavy chain results fromthe second round of RT-PCR are shown in FIG. 19, Panel D.

FIG. 20 shows a microfluidic device according to an embodiment of theinvention described herein, showing a reversible trap. (A) brightfieldimage at 20× magnification; (B) brightfield image at 40× magnification.

FIG. 21 shows a schematic whereby a microfluidic device according to anembodiment of the invention described herein is used as describedherein. (1) Flush chip with 1×PBS; (2) Load antibody-secreting cellsinto chambers; (3) Load antibody-capture beads into inlet channel; (4)Load antibody-capture beads into chamber against bead filter; (5)Incubate cells for 1 hour to allow antibody secretion and capture onbeads; (6) Wash out unbound antibody; (7) Load fluorescently-labeledantigen into inlet channel; (8) Flush chambers withfluorescently-labeled antigen; image and measure antibody-antigenassociation kinetics; (9) Flush our unbound antigen with 1×PBS; imageand measure antibody-antigen dissociation kinetics; and (10) Open sievevalve and flush cell out of the chamber to the elution port for recoveryfrom device.

FIG. 22 shows a schematic diagram of an alternative embodiment of themicrofluidic device for assaying binding interactions.

DETAILED DESCRIPTION

A binding interaction, as referred to herein, includes a molecularinteraction. A molecular interaction is commonly understood as referringto a situation when two or more molecules are attracted to one anotherby a force, where the force could be for example, electrostatic,dipole-dipole, hydrogen bonding, covalent, or hydrophobic in nature. Abinding affinity is commonly understood as referring to an averagestrength of a molecular interaction. Similarly, “avidity” is used todescribe the combined strength of multiple interactions. When used inthe present application, “affinity” is meant to encompass one or moreinteractions, including avidity. The methods described herein mayinvolve determining the binding affinity of the protein of interest. Themethods described herein may also involve determining a dissociationrate; and association rate and dissociation rate. Alternatively, themethods described herein may include determining binding kinetics.

The method may involve testing the antigen binding affinity byfluorescence imaging. The method may involve testing the antigen bindingaffinity using any of the following techniques plasmon resonance (SPR)spectroscopy, fluorescence anisotropy, or interferometry. Thesetechniques are understood to measure antibody-antigen binding kinetics,including, but not limited to surface plasmon resonance (SPR)spectroscopy, fluorescence anisotropy, interferometry, or fluorescenceresonance energy transfer (FRET). See, for e.g., Bornhop et al. (2007),Science 317: 1732-1736; Homola et al. (1999) Sensors and Actuators B:Chemical 54: 3-15; and Xavier, K. A. and Willson, R. C. (1998), Biophys.J. 74: 2036-2045. Further, the method may involve testing the antigenbinding affinity by a nanocalorimeter or a nanowire nanosensor. See, fore.g., Wang et al. (2005), Proc. Natl. Acad. Sci. USA 102: 3208-3212 andLee et al. (2009) Proc. Natl. Acad. Sci. USA 106: 15225-15230. Anothermethod that could be employed would be to use a technique such asdark-field microscopy and use antigens or antibodies labeled with goldnanoparticles. This could be used to detect single molecules andgenerate on/off rates by counting the molecules. See, for e.g., Ueno etal. (2010) Biophysical J. 98: 2014-2023; Raschke et al. (2003) NanoLetters 3: 935-938; and Sinnischen et al. (2000) App. Phys. Letters 77:2949-2951. Further methods for labeling and detecting binding eventsand/or binding kinetics would be known to a person of skill in the art.For example, binding assays may include determining the number ofbinding events.

A protein, as referred to herein, refers to organic compounds made ofamino acids, including both standard and non-standard amino acids.Standard amino acids include the following: alanine, cysteine, asparticacid, glutamic acid, phenylalanine, glycine, histidine, isoleucine,lysine, leucine, methionine, asparagine, proline, glutamine, arginine,serine, threonine, valine, tryptophan, and tyrosine. An example of aprotein is an antibody.

A biomolecule, as referred to herein, may include, but is not limitedto, an antibody, or an antibody fragment, or a whole cell, or a cellfragment, or a bacterium, or a virus, or a viral fragment, a nucleicacid or a protein.

A “chamber”, as used herein, refers to an enclosed space within amicrofluidic device in which a cells may be retained. Each chamber mayhave at least one inlet for permitting fluid, including fluid containinga cell, to enter the chamber, and at least one outlet to permit fluidand/or the cell to exit the chamber (depending on the design of thechamber and/or the flow through the chamber). Persons skilled in the artwill understand that an inlet or an outlet can vary considerably interms of structure and dimension, and may be characterized in a mostgeneral sense as an aperture that can be reversibly switched between anopen position, to permit fluid to flow into or out of the chamber, and aclosed position to seal the chamber and thereby isolate and retain itscontents. Alternatively, the aperture may also be intermediate betweenthe open and closed positions to allow some fluid flow or may be a sievevalve that allows for fluid flow out of the cell, but not otherparticles (for example, the cell, the beads etc.). A chamber, asreferred to herein, refers to a portion of a microfluidic device whichis designed to hold, for example, a cell. As used herein, the chamber isof an exceptionally small and discrete sizing. Typical volumes are inthe range of ˜100 pl to ˜100 nl. For example, a chamber can be designedwith a volume of approximately 500 pL (less than 1 nL), with dimensionsof approximately 100 microns (width), 500 microns (length), and 10microns (height).

The direction of fluid flow through the chamber dictates an “upstream”and a “downstream” orientation of the chamber. Accordingly, an inletwill be located at an upstream position of the chamber, and an outletwill be generally located at a downstream position of the chamber. Aperson skilled in the art will understand, however, that a singleaperture could function as both an inlet and an outlet.

An “inlet” or an “outlet”, as used herein, may include any aperturewhereby fluid flow is restricted through the inlet or outlet. There maybe a valve to control flow, or flow may be controlled by separating thechannels with a layer which prevents flow (for example, oil).Alternatively, an aperture may serve as both an inlet and outlet.Furthermore, an aperture (i.e. inlet or outlet) as used herein is meantto exclude the surface opening of a microwell.

A “microfluidic device”, as used herein, refers to any device thatallows for the precise control and manipulation of fluids in ageometrically constrained structure. For example, where at least onedimension of the structure (width, length, height) is less than 1 mm.

A solution, as referred to herein, may include, but is not limited to, asolution that can maintain the viability of a cell. Further, thesolution may include a suitable buffer that can both retain theviability of a cell such that binding interactions can be obtained orallow for an effective lysis of the cell to obtain nucleic acids fromthe cell and/or antibodies or other proteins depending on theapplication. Alternatively, the solution may be suitable for performingan assay.

A capture substrate, as referred to herein, is meant to encompass a widerange of substrates capable of capturing a protein or biomolecule ofinterest. These substrates may be modified to alter their surface(internal and external) properties depending on the desired use. Forexample, a substrate may be bound to antibodies or antigens to capturean antibody of interest. A capture substrate may be, for example, amicrosphere or a nanosphere or other microparticles including, but notlimited to a polystyrene bead or a silica bead (for example, antibodycapture beads and oligo(dT) mRNA capture beads). In an alternatearrangement, instead of modifying the beads with oligo(dT), specificprimers could be utilized instead. Optionally, the microsphere may be acarboxylic acid (COOH) functionalized bead. Beads which make use ofalternate chemical interactions can fall within this definition. See:for e.g., G. T. Hermanson (2008), Bioconjugate Techniques, 2nd Edition,Published by Academic Press, Inc. For example, an alternate scheme forpreparing these beads would be to use streptavidin coated beads and tomix these beads with biotinylized rabbit anti-mouse pAbs andbiotinylated oligo(dT). A capture substrate can also be an anti-Ig beadwhich binds an antibody to the capture substrate. A capture substratecan be modified such that it binds multiple biomolecules of interest,for example both mRNA and protein. Alternately, each capture substratecould be limited to a particular biomolecule, for example, one capturesubstrate being limited to binding mRNA and a second capture substratebeing limited to binding a protein. Capture substrates are commerciallyavailable or may be made de novo and/or modified as needed for theparticular application. Capture substrates may be removable, as in thecase of beads. However, capture substrates may also be fixed (and thus,non-removable).

Nucleic acids, as referred to herein, include macromolecules composed ofchains of monomeric nucleotides. Common examples of nucleic acidsinclude deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

In a further embodiment, a cell assay method is provided. The methodinvolves distributing an antibody producing cell (APC) to a chamber,wherein the APC is in a first solution, and wherein there is on averageone APC in the chamber; replacing the first solution with a secondsolution while maintaining the APC in the chamber; placing theantibodies produced by the APC in fluid communication with an antigen;and testing the binding of the antibodies produced by the APC with theantigen. Optionally, the method may involve adding anti-Ig beads to thechamber to capture the antibodies produced by the APC. Optionally, themethod may involve lysing the APC to capture antibodies produced by theAPC wherein the antibodies are not secreted by the APC.

A cell as referred to herein includes an antibody producing cell (alsoreferred to herein as an “APC”). An APC refers to a cell that canproduce an antibody. An antibody producing cell is not limited to cellsthat secrete antibodies, which are also referred to herein as antibodysecreting cells (also referred to herein as an “ASC”). For example, itwill be understood from the relevant art that memory B cells, withoutstimulation, do not normally secrete antibodies. See, for e.g., Abbas etal. (1997), Cellular and Molecular Immunology, 3^(rd) Ed., pp. 22-23).Examples of antibody producing cells (APCs) include B cells, memory Bcells, primary B cells (which are also known in the art as naïve Bcells), and B cell hybridomas. A primary B cell can be harvested fromthe spleen, blood, or bone marrow of an animal, for example from amouse, by FACS sorting for a cell surface marker, for example, theCD138+ marker (See: for e.g., Smith et al. (1996) Eur. J. Immunol. 26:444-448).

Antibodies are defense proteins produced by the vertebrate adaptiveimmune system for the purposes of binding and targeting for clearance adiverse range of bacteria, viruses, and other foreign molecules(antigens). As a result of their ability to bind target antigensselectively and with high affinity, antibodies are invaluable tools forprotein purification, cell sorting, and diagnostics. Antibodies areproduced by B cells and are secreted by activated B cells. (Seegenerally, for e.g., Abbas et al. (1997), Cellular and MolecularImmunology, 3^(rd) Ed., Chapter 3, pp. 37-65). Antibodies are alsoreferred to herein as immunoglobulin (also referred to herein as Ig). Anantibody, as referred to herein, can include, but is not limited topolyclonal antibodies and monoclonal antibodies. Unlike polyclonalantibodies, monoclonal antibodies are monospecific antibodies that arethe same because they are made by one type of immune cell that are allclones of a unique parent cell. A single APC or ASC can serve as thesource of a monoclonal antibody. Antibodies are not limited to aspecific isotype and can include, but are not limited to the followingisotypes: IgM, IgG, IgD, IgE, and IgA. Typically, it is understood thatantibodies are comprised of light and heavy chains that have variableand constant regions therein (see generally, for e.g., Abbas et al.(1997), Cellular and Molecular Immunology, 3^(rd) Ed., Chapter 3, pp.37-65).

In a further embodiment, a method of identifying a monoclonal antibodyof interest is provided. The method involves incubating an APC with aremovable capture substrate (RCS) in a suitable buffer, wherein theremovable capture substrate is capable of binding the monoclonalantibody produced by the APC and nucleic acids encoding the variableregions of the monoclonal antibody; and screening the bound removablecapture substrate to determine whether the APC produces the monoclonalantibody of interest.

In a further embodiment, a cell assay method is provided. The methodinvolves distributing an APC to a chamber, wherein there is on averageone APC in the chamber, wherein the APC is incubated with a removablecapture substrate in a first solution, and wherein the removable capturesubstrate is capable of binding an antibody of interest produced by theAPC and nucleic acids encoding the variable regions of the antibody ofinterest; replacing the first solution with a second solution whilemaintaining the APC in the chamber; placing the antibody of interestproduced by the APC in fluid communication with an antigen; andscreening the bound removable capture substrate to determine whether theAPC produces the antibody of interest.

In a further embodiment an apparatus for selecting a cell that producesa protein having a binding affinity for a biomolecule is provided. Theapparatus includes a microfluidic device operably configured to hold analiquot, wherein the aliquot on average contains one cell, and whereinthe protein produced by the cell is in fluid communication with thebiomolecule; and a detector for detecting the binding affinity of theprotein produced by the cell. However, the microfluidic device may alsohold more than one cell, particular in an assay where the antigen orbiomolecule of interest is a cell, or a cell fragment. Similarly, theantigen may be a virus or a bacterial cell.

In a further embodiment, a kit for identifying a cell that producesantibodies having a binding affinity for an antigen is provided. The kitincludes a microfluidic device and a removable capture substrate.

An antigen, as referred to herein, refers to a molecule recognized bythe immune system. As such, an antigen can include a molecule that canelicit an immune response in an organism, including in an animal.Examples of antigens include, but are not limited to bacterial antigensand viral antigens.

A method is provided for identifying antibody secreting cells (ASCs)that produce antibodies having a particular binding affinity for anantigen or functional attributes. The method involves distributing anASC within a discrete aliquot wherein there is on average one ASC in thealiquot, placing the antibodies in fluid communication with the antigen;and testing the antigen binding affinity of the antibodies produced bythe ASC. The method is based in part on the discovery that a single ASC,without clonal expansion, is capable of producing enough antibodies totest binding affinity for an antigen or to test other functionalattributes. Furthermore, the method is also based, in part, on thediscovery that clonal expansion via the production of hybridomas is notrequired for larger scale production of monoclonal antibodies, wherebythe variable regions for the antibodies of interest may be sequencedfrom an ASC of interest or collected with antibodies.

By way of example, a sensitive, low-cost microfluidic bead-basedfluorescence assay is described herein for measuring antibody-antigenbinding kinetics within low abundance samples. Direct measurements ofantibody-antigen binding kinetics may be made by time-coursefluorescence microscopy of antibody-conjugated beads retained inmicrofluidic chambers and subject to a series of wash cycles withfluorescently-labeled antigen and buffer. A variation of the bead-basedassay may include measuring the dissociation kinetics of unlabeledantibody and antigen molecules. As disclosed herein, multipleantibody-antigen interactions were measured spanning nearly four ordersof magnitude in equilibrium binding affinity. The rate constantsmeasured by way of the assay disclosed herein were validated withpreviously published values using SPR spectroscopy.

The methods provided herein are also contemplated for being used toscreen mutagenic B cell lines. Further, the methods provided herein arecontemplated for being used to screen the selectivity and specificity ofantibodies to multiple different antigens.

Antibody Binding Kinetics

The affinity or binding strength of an antibody for its target antigenis an important parameter when selecting an antibody for a givenapplication. Although the affinity of an antibody-antigen interaction istypically quantified by an equilibrium binding constant (K_(d)), whichdescribes the dynamic equilibrium between binding and unbinding events,the kinetic rate constants (k_(on) and k_(off)) provide a more completecharacterization of an antibody-antigen interaction. Two antibodies withidentical K_(d) values may exhibit dramatically different bindingkinetics which, in turn, will determine their respective suitability fora given application. For instance, antibodies with rapid association anddissociation kinetics may be desirable for sensing applications, whereasantibody-antigen interactions with very long half-lives may be criticalfor histological staining, enzyme-linked immunosorbent assays (ELISA),and Western blotting. Similarly, therapeutic antibodies that bind theirtarget antigens with long half-lives could, in principle, beadministered in lower dosages, reducing the cost and side-effects ofthese therapies. Direct measurement of binding kinetic constants can bea critical factor for selecting antibodies for both clinical andresearch applications. Examples of kinetic assays include, but are notlimited to viral and other pathogenic neutralization, cell signaling andgrowth inhibition, modulation of enzymatic activity (inhibit orenhance).

Microfluidics

Microfluidics refers to a multidisciplinary field dedicated to thedesign of systems in which small volumes of fluids will be used for avariety of purposes, including lab-on-a-chip technology. See: for e.g.,Squires and Quake (2005), Reviews of Modern Physics 77: 977-1026.Microfluidic technologies enable small-scale (picoliter to nanoliter)fluid handling operations for high-throughput biochemical analyses withlow reagent costs and rapid analysis times. In particular, microfluidicdevices fabricated from a silicone rubber, polydimethylsiloxane (PDMS),can be designed and fabricated in 24-48 hours, enabling rapidprototyping of devices. See: for e.g., McDonald, J. C. et al. (2000),Electrophoresis 21: 27-40. Microfluidic devices that integrate valvesinto pumps, mixers, fluidic multiplexers (MUXes), and otherfluid-handling components have been successfully applied for proteincrystallization, chemical synthesis, protein and DNA detection andsingle cell analysis. See, for e.g., Thorsen et al. (2002), Science 298:580-584; Hansen, et al. (2002), Proc. Natl. Acad. Sci. USA 99:16531-16536; Maerkl, S. J. & Quake, S. R. (2007) Science 315: 233-237;Hansen et al. (2004), Proc. Natl. Acad. Sci. USA 101, 14431-14436;Huang, B. et al. (2007) Science 315, 81-84; and Cai et al. (2006) Nature440: 358-362. Microfluidic devices, as described herein, can includechambers of varying sizes. For example, chambers can be designed with avolume of approximately 500 pL (less than 1 nL), with dimensions ofapproximately 100 microns (width), 500 microns (length), and 10 microns(height).

As disclosed herein, antibody-antigen binding kinetics were measuredwith approximately 4×10⁴ antibody molecules (˜66 zeptomoles) immobilizedon a single bead and less than 2×10⁶ antibodies (˜3 attomoles) loadedinto the microfluidic device. This represents a reduction of greaterthan four orders of magnitude in both detection limit and sampleconsumption compared to SPR spectroscopy and a recently reportedmicrofluidic fluorescence assay for measuring protein-protein bindingkinetics. See, for e.g., Bates, S. R.; Quake, S. R. (2009), Appl. Phys.Lett. 95, 073705. Since each antibody-antigen interaction can becharacterized on a single bead, millions of distinct antibody-antigeninteractions can be characterized with a single lot of commerciallyavailable beads (i.e., lmL at 10⁷-10⁸ beads/mL). By using the beadsurface rather than the chip surface as the sensor, a singlemicrofluidic device may be re-used indefinitely and may be imaged usinga standard inverted fluorescence microscope. However, a person of skillin the art could also apply the basic methods described herein to amicrofluidic system having antigen and/or antibodies bound to thesurface of a chip. It is further shown herein that an assay applying amethod described herein may be used to perform simultaneous kineticmeasurements of multiple antibody-antigen interactions using spatial andoptical multiplexing. By comparison, characterization of eachantibody-antigen interaction using SPR spectroscopy requires specializedinstrumentation and a unique flow cell on comparatively expensive sensorchips. The low detection limit of the microfluidic bead assay coupledwith small volume compartmentalization was exploited in order to measurethe antigen binding kinetics of antibodies secreted by a single ASC. Itis contemplated that the microfluidic bead assay described herein couldbe used for measuring antibody-antigen binding kinetics from rare bloodsamples, for screening scarce antibodies produced by primary plasmacells from immunized animals, as well as for selecting clones forrecombinant protein production. Additionally, it is contemplated that inaddition to its utility for measuring antibody-antigen binding kinetics,the microfluidic bead-based assay described herein can be used formeasuring other protein-protein and biomolecular interactions with awide range of binding affinities, such as protein-carbohydrate binding,protein-DNA (i.e., transcription factor binding) and protein-RNAinteractions. It is also contemplated that upon identifying an ASC thatsecretes antibodies which are optimal for a particular purpose, the ASCin question can be cloned by reverse-transcriptase PCR and standardizedcloning techniques.

Experimental Methods Microfluidic Device Fabrication and Control

All microfluidic devices were fabricated using multilayer softlithography (see, for e.g., Unger, M. A. et al. (2000), Science, 288:113-116 and Thorsen, T. et al. (2002), Science 298: 580-584. Deviceswere composed of two layers of poly(dimethylsiloxane) (PDMS) elastomer(GE RTV 615) bonded to No 1.5 glass coverslips (Ted Pella, Inc.). Thedevices were designed in AutoCAD software (Autodesk) and printed on highresolution (20,000 dpi) transparency masks (CAD/Art Services). Mastermolds were fabricated in photoresist on silicon wafers (Silicon Quest)by standard optical lithography. The control master molds werefabricated out of 20-25 m high SU-8 2025 photoresist (Microchem). Theflow master molds were fabricated with 12 m rounded SPR220-7.0photoresist channels (Rohm and Haas) and 6 μm SU-8 5 photoresist(Microchem) channels with rectangular cross-section. Microfluidic valveswere actuated at 30 psi pressure which was controlled using off-chipsolenoid valves (Fluidigm Corp) controlled using LabView 7.1 softwareand a NI-6533 DAQ card (National Instruments). Compressed air (3-4 psi)was used to push reagent solutions into the device.

Reagent Preparation

Protein A-coated 5.5 μm diameter polystyrene beads (Bangs Labs) wereincubated with lmg/mL solutions of Rabbit anti-mouse polyclonalantibodies (pAbs) (Jackson Immunoresearch). All antibody and antigensolutions were prepared in PBS/BSA/Tween solution consisting of 1×PBS,pH 7.4 (Gibco) with 10 mg/mL BSA (Sigma) and 0.5% Polyoxyethylene (20)sorbitan monolaurate (similar to Tween-20, EMD Biosciences). Lysozymefrom chicken egg white (HEL) was purchased from Sigma, and the D1.3 andHyHEL-5 mouse monoclonal antibodies to lysozyme were generously providedby Dr. Richard Willson (University of Houston). The anti-GFP mousemonoclonal antibody (LGB-1) was purchased from Abcam. Fluorescentprotein conjugates were prepared using Dylight488 and Dylight633 NHSesters (Pierce) and were purified using Slide-A-Lyzer dialysis cassettes(Pierce). The concentration of fluorescent conjugates was measured byspectrophotometry (Nanodrop). In order to minimize protein denaturation,fluorescent HEL conjugates were labeled at dye-to-protein (D/P) ratiosof less than 1, whereas the D1.3-Dylight488 conjugate was prepared at aD/P ratio of ˜5.

Microscopy

The microfluidic devices were imaged on a Nikon TE200 Eclipse invertedepifluorescence microscope equipped with green (470/40 nm excitation,535/30 nm emission) and red (600/60 nm excitation, 655 nm long-passemission) fluorescence filter cubes (Chroma Technology). Fluorescenceimages were taken using a 16-bit, cooled CCD camera (Apogee Alta U2000)and a 100× oil immersion objective (N.A. 1.30, Nikon Plan Fluor). Thesensitivity of the fluorescence measurements was tuned by binning pixelson the CCD detection camera and modulating the fluorescence exposuretimes (20 ms-1 s) with a computer-controlled mechanical shutter (Ludl).

Cell Culture

Mouse D1.3 hybridoma cells were cultured in RPMI 1640 media (Gibco) with10% FCS. Prior to loading into microfluidic devices, cells were washedby centrifugation at 1500 rpm and re-suspended in fresh media in orderto remove antibodies secreted in the cell media.

Microfluidic Bead-Based Fluorescence Assay

A microfluidic device was designed and fabricated to perform bead-basedfluorescence measurements of antigen-antibody binding kinetics (seeFIGS. 1A and B herein). The device consists of six fluidic inlets, eachused for loading a distinct reagent and controlled with an independentcontrol valve, which join into a common fluidic outlet. The fluidicoutlet can be partitioned into discrete ˜200 μL chambers by actuating aset of microfluidic “sieve” valves which, when actuated, act as filtersto immobilize large particles (>1 micron) while still allowing fluidexchange. See, for e.g., Marcus, J. S. et al. (2006) AnalyticalChemistry 78: 3084-3089.

At the start of the experiment, the fluidic outlet was flushed with aPBS/BSA/Tween solution from the top and bottom fluidic inlets in orderto pre-coat channel walls and reduce nonspecific binding. Next, 5.5 mdiameter Protein A beads coated with Rabbit anti-mouse pAb were loadedthrough the device to the fluidic outlet. The microfluidic sieve valveswere then actuated and the fluidic outlet was again washed withPBS/BSA/Tween solution to immobilize the beads against the traps andwash out any free rabbit pAb in solution. The beads were then washedwith the mouse antibody selected for kinetic characterization. Again,free mouse antibody was washed out of the fluidic outlet usingPBS/BSA/Tween. Finally, the beads were washed with fluorescently-labeledantigen and fluorescently imaged at defined time intervals to measurethe rate of antibody-antigen association (see FIG. 1C herein). Whenchemical equilibrium between the antibody and antigen was reached, asdetected by a plateau in bead fluorescence, the beads were flushed withPBS buffer and imaged to measure the rate of antibody-antigendissociation. The process was repeated with varying concentrations offluorescently-labeled antigen, each loaded onto the microfluidic devicefrom a separate fluidic inlet.

A second version of the microfluidic bead assay was implemented toindirectly measure dissociation kinetics between antibodies andunlabeled antigen molecules by displacement with fluorescently labeledantigen (see FIG. 1D herein). In this assay, after the antibody ofinterest was captured on Rabbit anti-mouse pAb-coated Protein A beads,beads were washed with unlabeled antigen at high concentration (>1 μM)to saturate all antibody binding sites. Beads were then washed withfluorescently labeled antigen while imaging at defined time intervals.Dissociation of the unlabeled antigen was then inferred by accumulatedfluorescence on the beads.

In order to measure the antigen binding kinetics from antibodiessecreted from single cells, Protein A beads coated with Rabbitanti-mouse pAb were first immobilized in the fluidic outlet channelusing the microfluidic sieve valves. A solution of RPMI-1640 mediacontaining 10⁵ hybridoma cells/mL was then loaded into the device from aseparate fluidic inlet and the control valve was momentarily opened toallow for a single hybridoma cell to be brought in close proximity withbeads immobilized in the first sieve trap in the fluidic outlet channel.The hybridoma cell was then allowed to incubate next to the beads for 1hour, and subsequently washed with PBS/BSA/Tween buffer to wash out anyfree antibody in solution and halt antibody secretion from the cell.Kinetic measurements of antigen binding were then performed in the samemanner as with purified antibodies.

FIG. 2 shows a schematic diagram of a microfluidic device operable fordetecting antibody secreted from antibody secreting cells. The stepsutilized are, for example, as follows: (1) flush microfluidic channelswith cell culture media; (2) load channels with antibody secreting cells(top) and capture beads (bottom); (3) incubate cells with beads tocapture secreted antibody; (4) trap cells and beads with sieve valvesand flush out unbound antibody by blowing buffer over cell-bead mixture;(5) flow fluorescently-labeled antigen over trapped cells and beads; and(6) flush out unbound antigen by blowing buffer over trapped cells andbeads and image fluorescent beads.

Data Analysis.

Fluorescent images were analyzed using MaximDL 4 imaging software.Fluorescent intensities were measured by selecting line profiles throughthe beads and recording the maximum intensity at the bead surface.During protein binding experiments, line profiles were constructedthrough the same beads at each measurement time point in order to avoidany systemic variations caused by differences in bead-to-bead bindingcapacity, variation in position in the flow channel and non-uniformillumination over the field of view. The measured fluorescence beadintensities were assumed to be proportional to the concentration ofantibody-antigen complex ([AbAg]) and were fit to the followingfirst-order, mass action and Langmuir isotherm equations using nonlinearleast squares minimization:

$\begin{matrix}{{F(t)} = {{\left( {F_{\max} - F_{0}} \right)\frac{\lbrack{Ag}\rbrack_{o}}{\lbrack{Ag}\rbrack_{o} + K_{d}}\left( {1 - e^{{- {({{k_{on}{\lbrack{Ag}\rbrack}}_{0} + k_{off}})}}t}} \right)} + F_{0}}} & \left( {{equation}\mspace{14mu} 1} \right) \\{{F(t)} = {{\left( {F_{\max} - F_{0}} \right)\frac{\lbrack{Ag}\rbrack_{o}}{\lbrack{Ag}\rbrack_{o} + K_{d}}e^{{- k_{off}}t}} + F_{0}}} & \left( {{equation}\mspace{14mu} 2} \right) \\{{F(t)} = {{\left( {F_{\max} - F_{0}} \right)\frac{\lbrack{Ag}\rbrack_{o}}{\lbrack{Ag}\rbrack_{o} + K_{d}}} + F_{0}}} & \left( {{equation}\mspace{14mu} 3} \right)\end{matrix}$

where F(t) represents the measured bead fluorescence at time t, F₀ andF_(max) represent the background and maximum bead fluorescence,respectively, [Ag]₀ represents the solution concentration of antigen (inM), and, k_(f) and k_(r) represent the intrinsic association anddissociation rate constants, in units of M-¹s⁻¹ and s⁻¹, respectively.

EXAMPLES

The following examples describe embodiments of the invention detailedherein.

Example 1. Measurement of Antibody-Antigen Binding Kinetics on Beads

The binding kinetics of the D1.3 mouse monoclonal antibody (mAb) tofluorescently-labeled hen egg lysozyme (HEL) was measured using themethodologies and techniques described herein. See: FIG. 3A and Table 1herein.

TABLE 1 Antibody-antigen binding kinetics measured using themicrofluidic fluorescence bead assay. Antigen interaction Antibodyk_(on) (M⁻¹s⁻¹) k_(off) (s⁻¹) K_(d) D1.3 mAb/HEL-Dylight488 1.87 ± 0.48× 10⁶ 2.10 ± 0.25 × 10⁻³ 1.20 ± 0.42 nM D1.3 mAb/HEL-Dylight633 1.27 ±0.22 × 10⁶ 2.15 ± 0.23 × 10⁻³ 1.75 ± 0.46 nM HyHEL-5 mAb/HEL-Dylight6335.75 ± 0.71 × 10⁶ 1.69 ± 0.30 × 10⁻⁴  30.0 ± 7.4 pM LGB-1 mAb/EGFP 5.00± 0.72 × 10⁴ 5.15 ± 0.89 × 10⁻³  106 ± 28 nM

The measured association and dissociation rate constants for theD1.3/HEL interaction were 1.87±0.48×10⁶ M⁻¹s⁻¹ and 2.10±0.25×10⁻³ s⁻¹,respectively, and were consistent with values of 1.0-2.0×10⁶ M⁻¹s⁻¹ and1.15-3.04×10⁻³ s⁻¹ previously measured using surface plasmon resonance(SPR) spectroscopy, stopped-flow fluorescence quenching, and competitiveELISA. See, for e.g., Batista, F. D. and Neuberger, M. S. (1998),Immunity 8: 751-759 and Ito, W. et al. (1995), Journal of MolecularBiology 248: 729-732. A ten-fold smaller association rate constantpreviously reported for the D1.3/HEL interaction (1.67×10⁵ M⁻¹s⁻¹) canlikely be attributed to differences between the full D1.3 mAb used inour microfluidic bead-based measurements and the recombinantsingle-chain antibody fragment used by Bedouelle and coworkers (Englandet al. (1999) J. Immunol. 162: 2129-2136).

Additionally, indirect, label-free measurements of the D1.3 mAb/HELdissociation rate constant using a variation of our microfluidic beadassay were performed using the methodologies and techniques describedherein. See: FIGS. 1D and 3D herein. In this assay, D1.3 mAbsimmobilized on beads were first saturated with unlabeled HEL andsubsequently washed with fluorescently-labeled HEL. Measurements of theaccumulated bead fluorescence faithfully reflected the D1.3/HELdissociation kinetics provided the labeled HEL was at a sufficientlyhigh concentration to ensure that dissociation was rate-limiting (i.e.k_(on)[Ag]>k_(off), or, equivalently, [Ag]>K_(d)). Using this method,the dissociation rate constant of D1.3 and unlabeled HEL was measured tobe 1.45±0.30×10⁻³ s⁻¹, in close agreement with direct dissociationmeasurements between D1.3 and fluorescently-labeled HEL. See Table 1herein.

The microfluidic bead assay was used to measure the binding kinetics ofHEL and HyHEL-5, a distinct mouse mAb with significantly strongerbinding affinity to HEL than D1.3. In comparison to the D1.3 mAb,HyHEL-5 bound HEL with a nearly four-fold larger association rateconstant (5.75±0.71×10⁶ M⁻¹s⁻¹) and ten-fold smaller dissociation rateconstant (1.69±0.30×10⁻⁴ s⁻¹). See: FIG. 3B herein. Thus, HyHEL-5 boundHEL with a ˜40-fold smaller equilibrium dissociation constant than D1.3(30 pM vs. 1.2 nM). See: Table 1 herein. Compared with the microfluidicbead assay, previous measurements of the HyHEL-5/HEL interaction usingsolution-phase fluorescence anisotropy resulted in a similardissociation rate constant (2.2×10⁻⁴ s⁻¹), but a three- to five-foldlarger association rate constant (1.5-3.3×10⁷ M⁻¹s⁻¹). See, for e.g.,Xavier, K. A. and Willson, R. C. (1998) Biophys. J. 74: 2036-2045. SinceHyHEL-5 mAb binds HEL with near diffusion-limited association kinetics,immobilization of the mAb in the microfluidic bead assay couldpotentially result in slower association kinetics when compared withsolution-phase fluorescence anisotropy measurements. However, since thediffusion constant of HEL is approximately three times larger than thatof the mAb, immobilization of the HyHEL-5 mAb would reduce the effectivediffusion coefficient (D≅D_(mAb)+D_(HEL)) and, hence, the apparentassociation rate constant by at most 25%. See, for e.g., Tyn, M. T. andGusek, T. W. (1990), Biotechnology and Bioengineering 35: 327-338 andHe, L. and Niemeyer, B. (2003), Biotechnol. Prog. 2003, 19: 544-548.Therefore, the difference in measured and reported association rateconstants is likely a result of different buffer solutions, as theHyHEL-5 and HEL binding interaction is known to be very sensitive tosolution pH and buffer salt concentration. See, for e.g., Xavier, K. A.and Willson, R. C. (1998) Biophys. J. 74: 2036-2045 and Dlugosz et al.(2009), The Journal of Physical Chemistry 113: 15662-15669.

The binding kinetics of a commercially available mouse monoclonalantibody (LGB-1, Abcam) to enhanced green fluorescent protein (eGFP)were also measured using the methodologies and techniques describedherein. See: FIG. 3 herein. This binding interaction was chosen todemonstrate that the bead-based assay can be used to measure bindingkinetics of a previously uncharacterized antibody without optimizing thebead immobilization chemistry. In this instance, native eGFPfluorescence was measured without an exogenous fluorescent label. Themeasured association and dissociation rate constants for the LGB-1/eGFPinteraction were 5.00±0.72×10⁴ M⁻¹s⁻¹ and 5.15±0.89×10⁻³ s⁻¹,respectively. See: Table 1 herein.

Collectively, the measured binding kinetics of the anti-lysozyme andanti-eGFP mAbs span nearly four orders of magnitude in equilibriumdissociation constants (30 pM-100 nM), with association rate constantsvarying from 5×10⁴-10⁶ M⁻¹s⁻¹ and dissociation rate constants rangingfrom 10⁻³-10⁻⁴ s⁻¹. See: Table 1 herein. In principle, the microfluidicbead-based assay can be used to characterize stronger antibody-antigeninteractions than the HyHEL-5/HEL interaction; however, bindinginteractions with dissociation rate constants lower than 10⁻⁴ s⁻¹require measurements to be taken over several days or weeks. On theother hand, the bead-based assay can be readily used to measure bindinginteractions weaker than the LGB-1/eGFP interaction. Using this assay,the practical upper limit in measurable dissociation rate constants isapproximately 10⁻¹ s⁻¹, as a result of the time required to exchangesolutions in the microfluidic device. Thus, the microfluidic bead-basedassay should enable characterization of antibody-antigen interactionsthat span greater than six orders of magnitude in equilibrium bindingaffinity.

Example 2. Simultaneous Measurement of Multiple Antibody-Antigen BindingKinetics Using Optical and Spatial Multiplexing

The binding kinetics of multiple antibody-antigen interactions weremeasured simultaneously using both optical and spatial multiplexing ofthe bead-based assay using the methodologies and techniques describedherein. Each antibody was immobilized on a distinct population of beadsand, subsequently, beads from each population were sequentially trappedusing sieve valves on the microfluidic device. Since beads trapped bythe sieve valves remain immobilized throughout the duration of eachexperiment, the spatial address of beads was tracked in order toidentify each antibody. Subsequently, the trapped beads were washed witha mixture of antigens, each labeled with a spectrally distinctfluorophore. The beads were then imaged with different fluorescencefilter sets designed to coincide with each fluorescent antigen. In thismanner, the binding kinetics of 3 different monoclonal antibodies (D1.3,HyHEL-5 and LGB-1) to two different fluorescent antigens (HEL-Dylight488and eGFP) were simultaneously measured. See: FIG. 4 herein. By employingthis strategy, it was possible to spectrally distinguish which beadswere coated with anti-lysozyme mAbs or anti-eGFP mAbs, whereas the twoanti-lysozyme mAbs (D1.3 and HyHEL-5) were discriminated based on theirunique binding kinetics for HEL. In addition, the fluorescenceintensities of HyHEL-5 coated beads were significantly higher than theD1.3 coated beads, consistent with the fact that HyHEL-5 binds HEL witha significantly lower equilibrium dissociation constant than D1.3. See:FIG. 4 herein. This technique can be extended to measure any combinationof mxn antibody-antigen interactions in which m antibodies areimmobilized on different beads and exposed to a solution of n antigens,each with a spectrally-resolvable fluorescent label. In practice,several hundred antibody-antigen interactions could be measuredsimultaneously by imaging up to 100 beads in a single field of view withfive to six spectrally distinct fluorophores. Multiplexed beadmeasurements could be used for simultaneously analyzing the bindingkinetics and binding specificities of a panel of mAbs to multipledifferent antigens in serum and other complex mixtures.

Example 3. Microfluidic Bead-Based Fluorescence Measurements ReflectIntrinsic Antibody-Antigen Binding Kinetics

A series of experiments were performed using the methodologies andtechniques described herein to verify that bead-based fluorescencemeasurements reflected intrinsic antibody-antigen binding kinetics, andwere unaffected by artifacts arising from fluorescent labeling of theantigen, antibody immobilization, diffusion limitation or mass transporteffects. Fluorescent labeling of HEL did not alter the intrinsicD1.3/HEL binding kinetics, as indicated by the agreement betweenmicrofluidic bead-based measurements using fluorescently labeled HEL andpreviously reported measurements using SPR spectroscopy with unlabeledHEL. See, for e.g., Batista, F. D. and Neuberger, M. S. (1998), Immunity8: 751-759 and Ito, W. et al. (1995), Journal of Molecular Biology 248:729-732. Moreover, no differences were observed in bead-based kineticmeasurements of the D1.3 mAb binding to HEL labeled with two differentfluorophores, Dylight488 and Dylight633 (Pierce). See: Table 1 herein.It was ensured that photobleaching of fluorophores did not affect themeasured binding kinetics by measuring the photobleaching rates of theof the fluorescent dyes used in this study (Dylight488, Dylight633, andeGFP) and selecting fluorescence exposure times of less than 100 ms,such that each measurement resulted in less than 5% reduction in beadfluorescence. See: FIG. 7A herein. Indeed, measured binding kineticswere consistent over a large range of fluorescence exposure times (ms),whereas exposure times of greater than is resulted in substantialphotobleaching and an artificial increase in measured association anddissociation binding kinetics when compared to intrinsic kinetics. SeeFIG. 7B herein.

To examine the effect of different antibody bead immobilizationchemistries, we verified that measured association and dissociation rateconstants for the D1.3/HEL interaction were the same when captured onsilica or polystyrene beads coated with either rabbit or goat anti-mousepolyclonal antibody. See: FIG. 8 herein. It was further verified thatmultivalent binding between the rabbit anti-mouse pAbs andfluorescently-labeled D1.3 mAb resulted in no detectable dissociationover the course of 3 days, which would otherwise artificially acceleratethe measured antibody-antigen binding kinetics. See: FIG. 9 herein. Thenearly irreversible bond between rabbit pAb and the mouse mAbs wascritical to successful antibody-antigen binding kinetic measurements asattempts to measure D1.3/HEL binding kinetics using Protein A beadswithout Rabbit anti-mouse pAbs were unsuccessful due to rapiddissociation (and low affinity) of protein A/mouse mAb complexes.

Several experiments were also conducted to verify that diffusionlimitation and mass transport did not affect bead-based measurements ofantibody-antigen binding kinetics. In the diffusion-limited regime,antibodies adjacent on the bead surface would compete for fluorescentantigen, thus reducing the apparent association rate constant.Similarly, the apparent rate of antibody-antigen dissociation would bereduced due to antigen rebinding to adjacent antibodies. See, for e.g.,Berg, H. C. and Purcell, E. M. (1977), Biophys. J. 20: 193-219 andLauffenburger, D. A. and Linderman, J. (1965) Receptors: Models forBinding, Trafficking, and Signaling; Oxford University Press. Nearlyidentical association and dissociation kinetics for the D1.3-HELinteraction was measured by varying the amount of bead-immobilized D1.3mAb over two orders of magnitude. See FIG. 10B herein. Dissociationkinetics of the D1.3 antibody and fluorescently labeled HEL were alsosimilar both in the presence and absence of a high concentration (˜2mg/mL) of competitive unlabeled HEL antigen. See: FIG. 10 herein. Thus,there was no observable competition between antibodies adjacent to oneanother on the beads and, hence, no diffusion limitation. It was alsoconfirmed that the association and dissociation rate constants of theD1.3-HEL interaction remained constant over a range of flow rates from3-15 μL/hr, suggesting no effect of mass transport on the measuredkinetics. See: FIG. 11 herein.

Example 4. Bead-Based Kinetic Measurements Exhibit Low Detection Limitsand Minimal Sample Consumption

To quantify the detection limit and minimal sample consumption requiredfor microfluidic bead-based measurements of antibody-antigen bindingkinetics, antibody-antigen binding kinetics were measured using varyingamounts of bead-immobilized mAb along with the methodologies andtechniques described herein. The association rate constant offluorescently-labeled D1.3 mAb binding to Rabbit anti-mouse pAb coatedProtein A beads was measured. See: FIG. 5A herein. Using the measuredkinetic on-rate constant for this interaction (k_(on)=1.10±0.11×10⁶M⁻¹s⁻¹) and modulating the loading time of D1.3 mAb, the amount ofbead-immobilized D1.3 mAb was varied over two orders of magnitude. Then,the antibody-antigen binding kinetics with as little as 1% of the beadsurface covered with D1.3 mAb was successfully measured. See: FIG. 5Bherein. Using the manufacturer's specifications as well as stericconsiderations, a single 5.5 micron bead can bind 4×10⁶ antibodymolecules (˜6.6 amol); therefore, it was estimated that the detectionlimit of our microfluidic fluorescence bead assay is to be ˜4×10⁴antibodies or ˜66 zeptomoles. See: FIG. 5C herein. In contrast, SPRspectroscopy requires at least 200 pg (˜109 molecules) of immobilizedantibody in order to generate a detectable refractive index change. See,for e.g., Biacore Life Sciences—Biacore 3000 System Information.Website:http://www.biacore.com/lifesciences/products/systems_overview/3000/system_information/in dex.html. Additionally, D1.3/HEL binding kineticswere successfully measured by loading less than 2 million D1.3 mAbmolecules (˜3 attomoles) into the microfluidic device. In theory, theminimum sample consumption of the microfluidic bead assay could bereduced even further by reducing losses associated with channel deadvolumes and optimizing the capture efficiency of antibodies on beads, aswell as using microfluidic pumps to achieve flow rates less than 1μL/hr. Thus, when compared with alternative techniques and SPRspectroscopy, our microfluidic bead-based assay can measureantigen-antibody binding kinetics with a reduction in both detectionlimit and sample consumption by four orders of magnitude.

Example 5. Measurement of Binding Kinetics of Antigen and AntibodySecreted from Single Cells

Based on the low detection limit of the bead-based assay and in aneffort to measure the antigen binding kinetics of antibodies secretedfrom single antibody secreting cells, single D1.3 hybridoma cells wereloaded adjacent to Rabbit anti-Mouse pAb coated Protein A beads capturedin the microfluidic device, and then co-incubated the cells and beadsfor 1 hour at room temperature. See: FIG. 6 herein. Subsequently,antibody-antigen binding kinetics were measured by recording thefluorescence of a single bead washed with buffer and successively higherconcentrations of fluorescent antigen, in a manner analogous to thesingle-cycle kinetics technique used with SPR spectroscopy. See, fore.g., Biacore Life Sciences—Single-Cycle Kinetics. Website:http://www.biacore.com/lifesciences/technology/introduction/data_interaction/SCK/index.htm1 and Abdiche et al. (2008) Analytical Biochemistry 377: 209-217. Usingthe methodologies and experimental techniques described herein, theassociation and dissociation rate constants for the D1.3/HEL interactionwere successfully measured using antibodies secreted by a single D1.3hybridoma cell, which were consistent with measurements on purifiedantibodies. See: FIG. 6 and Table 1 herein.

Antibody-secreting cells are known to secrete thousands of antibodiesper second at 37° C., and would, therefore, secrete enough antibodies inapproximately one hour to saturate the surface of a single 5.5 m beadwith maximum binding capacity of ˜4×10⁶ antibody molecules. See, fore.g., Niels Jerne (1984) The Generative Grammar of the Immune System andMcKinney et al. (1995) Journal of Biotechnology 40: 31-48. While it isreasonable to suspect that the hybridoma cells secrete antibodies at areduced rate when incubated at room temperature; nonetheless, singleD1.3 hybridoma cells secreted sufficient antibody within 1 hour at roomtemperature for complete kinetic characterization. However, based on theincubation time and detection limit of the assay (˜4×10⁴ antibodies), itcan be inferred that single hybridoma cells secreted greater than 10antibodies/second when incubated at room temperature in the microfluidicdevice.

Examples 1-5 show that the methods described herein are suitable formeasuring antibody-antigen kinetics in a microfluidic environment from asingle cell.

Example 6. Dual Function Beads

An overview of a scheme for preparing dual-capture (i.e., dual function)beads using carbodiimide chemistry is shown in FIG. 14. A representativeexperiment utilizing dual function beads is shown in FIG. 15. Briefly,polystyrene COOH beads (Bangs Labs) were conjugated with Rabbitanti-mouse pAb (Jackson ImmunoResearch) and amine-functionalizedoligo(dT)₂₅ (Genelink) using carbodiimide chemistry. In FIG. 14(A), abrightfield image of dual-function beads trapped using a microfluidicsieve valve is shown. In FIG. 14(B), a fluorescence image of syntheticsingle-stranded DNA molecules captured on dual-function beads is shown.Synthetic DNA molecules are labeled with Cy5 fluorophore forvisualization and also contain a poly(A) tail that binds to theoligo(dT) on the bead surface. In FIG. 14(C), a fluorescence image ofmouse D1.3 monoclonal antibody (mAb) captured on dual-function beads.D1.3 mAbs are labeled with Dylight488 fluorophore for visualization andbind to the Rabbit anti-Mouse pAb on the bead surface.

It will be understood that while carboxylic acid (COOH) beads aredisclosed herein, other beads, which make use of alternate chemicalinteractions, could also be used, See: for e.g., G. T. Hermanson (2008),Bioconjugate Techniques, 2nd Edition, Published by Academic Press, Inc.For example, an alternate scheme for preparing the beads would be to usestreptavidin coated beads and to mix these beads with biotinylizedrabbit anti-mouse pAbs and biotinylated oligo(dT).

Example 7. Multiplex RT-PCR of the Antibody Heavy and Light Chain Genes

Results from a multiplex RT-PCR of the antibody heavy and light chaingenes indicated that a gene product coinciding with the proper molecularsize was obtained. Briefly, D1.3 hybridoma cells were lysed using anonionic detergent (1% NP-40 in 1×PBS) and the lysate was then mixedwith rabbit anti-mouse pAb, oligo(dT)-conjugated dual-capture beads formRNA capture. Generally, a gentle lysis buffer is preferred for celllysis and can include, in addition to the foregoing: 0.5% NP-40 in 1×PBSor DI water or 0.5% Tween-20 in 1×PBS or DI water. Generally, it ispreferable and within the knowledge of those persons skilled in the artto use lysis buffers that can sufficiently lyse the outer membrane ofthe cell in question, while keeping the nucleus intact. RT-PCR wasperformed using degenerate primers for both heavy and light chain genesand resulted in bands of the expected size for antibody heaby and lightchains (date not shown). The results suggest that dual purpose RNA andantibody beads are capable of capturing RNA suitable for amplification.For comparison, RT-PCR of antibody genes was performed usingcommercially available oligo(dT) beads and dual-capture beads. Themethodology utilized herein is generally as follows:

-   -   1) Capture oligo(dT) beads in microfluidic chambers using sieve        valves.    -   2) Load cells in microfluidic chambers.    -   3) Load antibody-capture beads in chambers.    -   4) Incubate cells and beads.    -   5) Measure antibody-antigen binding kinetics.    -   6) Lyse cells using either a) 1% NP-40 in 1×PBS, or b) alkaline        lysis solution (100 mM Tris-HCl, pH 7.5, 500 mM LiCl, 10 mM        EDTA, pH 8.0 1% LiDS, 5 mM dithiothreitol (DTT)). During lysis,        the cell lysate is flushed over the stack of trapped oligo(dT)        beads. The oligo(dT) beads and alkaline lysis solution are from        the Dynabead mRNA direct kit developed by Invitrogen, but        alternatives to these reagents exist.    -   7) Wash beads with 1×PBS to remove lysis solution.    -   8) Open sieve valves.    -   9) Open microfluidic chamber valves and send beads to an output        port (one chamber at a time).    -   10) Recover beads from output port using a pipette.    -   11) Pipette beads into 50 microL of one-step RT-PCR mix.        -   i) dNTPs;        -   ii) mixture of RT and DNA polymerase enzymes;        -   iii) degenerate primers for both heavy and light chain genes            (PCR reagents from a One-Step RT-PCR kit developed by            Qiagen, but could also be prepared ourselves);    -   12) Perform RT and Touchdown PCR using the following protocol:        -   i) RT at 50° C. for 30 min;        -   ii) 95° C. for 15 min to inactivate RT enzyme and activate            DNA polymerase        -   iii) First ten cycles of Touchdown PCR:        -   a) 94° C. for 30 s;        -   b) 55° C. for lmin (decrease by 1 C each cycle, until 45 C);        -   c) 72° C. for 1 min.        -   iv) 30 cycles of PCR        -   a) 94° C. for 30 s;        -   b) 45° C. for 1 min;        -   c) 72° C. for 1 min.    -   13) Visualize RT-PCR amplicons on 0.5% DNA agarose gel using        SYBRsafe fluorescent dye.    -   14) Extract amplicons from gel and purify using standard gel        extraction kit (Qiagen).    -   15) Sequence samples.

Example 8. Microfluidic Antibody-Antigen Binding Kinetics Measured UsingDual Function Beads and Antibodies Secreted by Single Hybridoma Cells

Microscope image of D1.3 hybridoma cell adjacent to Rabbit anti-MousepAb, oligo(dT)-conjugated polystyrene beads trapped by a microfluidicsieve valve. After a 2 hour incubation the beads with the D1.3 cell,antibody-antigen binding kinetics were measured using fluorescentlylabeled HEL-Dylight488 conjugate. These results are highlighted in FIG.16 and show that dual purpose beads are suitable for testingantibody-antigen binding kinetics.

Example 9. Mouse Experiment: Antibody Binding Kinetics and Whole-CellHeavy Chain RT-PCR with Beads

These experiments were designed to detect antibodies from primarysplenocytes harvested from BALB/c immunized mice. The cells were elutedand whole-cell single-plex RT-PCR was performed of heavy and light chainantibody genes. Thereafter, binding kinetics of the antibodies weremeasured.

Chip.

Bead v6.6 chip with ˜3 micron high sieve channels, 2 micron gratings(fabricated: May 29, 2011 with RTV615).

Reagents.

The following reagents were used herein: 1×PBS for reagent flush;FACS-sorted CD138+ primary splenocytes in RPMI-10-2-ME media; 4.9 micronRabbit anti-Mouse Protein A beads; 5 microL of stock bead solutionresuspended in 100 μL of RPMI-10-2-ME media; and 214 ng/mL HEL488 in1×PBS.

Experimental Protocol.

The following experimental protocol was followed: washed chip with1×PBS; closed sieve valves; spun down primary cells and decanted ˜400 of500 μL of media; and re-suspended cells in remaining media.

Thereafter, the cells were loaded in all chambers sequentially withdeliberate negative controls included (e.g., R1C14 and R0C02); load 4.9micron Rabbit anti-Mouse Protein A beads in all chambers sequentially;incubate cells and beads for 1 h 20 min; and wash all chambers with 214ng/mL HEL488 for 5 min. Thereafter, chamber intensities were analyzedusing Image Analysis. Positive chambers were determined as follows:R0C04, R0C08, R2C02, R2C04, R2C07, R2C12, R2C13, R3C06, R4C01, R4C03,R4C05, R5C08, R5C09, R5C12, R5C14, R6C01, R6C02, R6C09, R6C10, R6C11,and R7C05.

RT-PCR mix without primers was prepared from a One-Step RT-PCR kit(Qiagen). The mix comprised: 10 μL 5×RT-PCR buffer×16=160 μL; 21 μLRNase-free water×16=336 μL; 2 μL dNTPs×16=32 μL; 2 μL Enzyme mix×16=32μL. The RT-PCR mix without primers was aliquoted into 8 tubes of 70 μLeach (2 reaction volumes, not including primer volume).

Prepared primer solutions to be mixed with RT-PCR after cell elutionwere as follows: Heavy chain—7.5 μL 8 μM 3′ IgH first primer×8=60 μL;and 7.5 μL 8 μM 5′ IgH first primer×8=60 μL. Kappa chain—7.5 μL 8 μM 3′IgK first primer×8=60 μL; and 7.5 μL 8 μM 5′ IgK first primer×8=60 μL.15 μL of primer mixes were aliquoted into each of 8 tubes. The primersused herein were selected based on what is taught in Table II of Tilleret al. (2009) J. Immunol. Methods 350: 183-193, which is incorporatedherein by reference. Those persons skilled in the art that variants tothe primers defined in Table II could be used under certaincircumstances including, for example, the primers in Table III in Tilleret al. (2009).

Eight (8) of the brightest chambers [R0C04, R2C04, R2C07, R3C06, R5C12,R5C14, R6C01, R6C10] were eluted. The eluted cell samples were pipetteddirectly into 70 μL RT-PCR mix without primers. The RT-PCR/cell mix wassplit into two (2) equal parts of 35 μL and mixed with kappa and heavychain primers, respectively. RT-PCR was performed using a thermalcycler. Briefly, the “NEST1ST5” protocol was used for the kappa chainreactions, comprising: RT step: 50° C. for 30 min; Hotstart/RTinactivation: 95° C. for 15 min; and 50 Cycles (denaturation: 94° C. for30 s; anneal: 50° C. for 30 s; and extension: 72° C. for 55 s). Then,there was a final extension: 72° C. for 10 min. Heavy chain reactionsperformed using the “NEST1H” protocol, comprising: RT step: 50° C. for30 min; Hotstart/RT inactivation: 95° C. for 15 min; and 50 Cycles(denaturation: 94° C. for 30 s; anneal: 56° C. for 30 s; extension: 72°C. for 55 s). Then, there was a final extension: 72° C. for 10 min.

Thereafter, kinetics were measured on each of the eluted chambers. Asummary of the kinetics data is shown in FIG. 17. Representativekinetics sample data is shown in each of FIGS. 18A-E herein.Dissociation kinetics were measured and then association kinetics weremeasured using freshly loaded HEL488. Thereafter, a second round ofsingle-plex RT-PCR was performed using nested second round primers. Theheavy chain mix comprised of: 10 μL 5×RT-PCR buffer×8=80 μL; 21 μLRNase-free water×8=168 μL; 2 μL dNTPs×8=16 μL; 2 μL Enzyme mix×8=16 μL;7.5 μL 8 μM 3′ IgH second primer×8=45 μL; and 7.5 μL 8 μM 5′ IgH secondprimer×8=45 μL. The kappa chain mix comprised of: 10 μL 5×RT-PCRbuffer×8=80 μL; 21 μL RNase-free water×8=168 μL; 2 μL dNTPs×8=16 μL; 2μL Enzyme mix×8=16 μL; 7.5 μL 8 μM 3′ IgK second primer×8=45 μL; and 7.5μL 8 μM 5′ IgK second primer×8=45 μL. Thereafter, 3.5 μL of templatefrom each of the first round reactions was added and RT-PCR wasperformed on a thermal cycler. Kappa chain reactions were performedusing the “NEST2K” protocol. Briefly, there was no RT step; hotstart/RTinactivation was at 95° C. for 15 min followed by 50 cycles(denaturation: 94° C. for 30 s; anneal: 45° C. for 30 s; and extension:72° C. for 55 s). Then, there was a final extension: 72° C. for 10 min.

Heavy chain reactions performed using the “NEST2H” protocol. Briefly,there was no RT step; hotstart/RT inactivation: 95° C. for 15 min,followed by 50 cycles (denaturation: 94° C. for 30 s; anneal: 60° C. for30 s; and extension: 72° C. for 55 s). Then, there was a finalextension: 72° C. for 10 min. The RT-PCR products for both first andsecond round reactions were run on a gel. Kappa chain results from thefirst round are shown in FIG. 19A. Kappa chain results from the secondround are shown in FIG. 19B. Heavy chain results from the first roundare shown in FIG. 19C. Heavy chain results from the second round areshown in FIG. 19D. The gel products were sequenced by standardprocedures known to those skilled in the art. Based on the sequence datagenerated, variants in antibody sequences were detectable. As arepresentative example, mutations in the R00C04 sample are shown inTable 2 herein.

TABLE 2 R00C04 (9 non-synonymous mutations) Position (from Situation(from Germline Ab R00C04 Ab IMGT) IMGT) residue residue L-36 CDR1-L S NL-92 FR3-L S T H-17 FR1-H A D H-36 CDR1-H S R H-40 FR2-H H L H-64 CDR2-HN K H-65 CDR2-H T S H-83 FR3-H S I H-94 FR3-H P L

Example 10. Microfluidic Device

A microfluidic device has been developed for assaying a bindinginteraction between a protein produced by a cell and a biomolecule. Thedevice has a chamber having an aperture and a channel for receiving aflowed fluid volume through the chamber via said aperture. The channelprovides for size selection for a particle within the fluid volume.Alternately, another embodiment of the microfluidic device has a chamberhaving an aperture and a reversible trap. The reversible trap has spacedapart structural members extending across the chamber. The structuralmembers are operable to allow a fluid volume to flow through the chamberwhile providing size selection for a particle within the fluid volume.See, for example: FIG. 20 herein.

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. Numeric ranges areinclusive of the numbers defining the range. The word “comprising” isused herein as an open-ended term, substantially equivalent to thephrase “including, but not limited to”, and the word “comprises” has acorresponding meaning. As used herein, the singular forms “a”, “an” and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a thing” includes more thanone such thing. Citation of references herein is not an admission thatsuch references are prior art to the present invention. The inventionincludes all embodiments and variations substantially as hereinbeforedescribed and with reference to the examples and drawings. Further,citation of references herein is not an admission that such referencesare prior art to the present invention nor does it constitute anyadmission as to the contents or date of these documents.

1.-45. (canceled)
 46. A method of assaying for a binding interactionbetween an antibody secreted by a single antibody secreting cell (ASC)and an antigen, the method comprising: retaining the single ASC within achamber having a volume of from 100 μL to 100 nL, a solid wall, and anaperture that defines an opening of the chamber; incubating the singleASC within the chamber to produce a secreted antibody; bringing a firstfluid volume comprising the antigen in fluid communication with thesecreted antibody; exposing the secreted antibody to a removeablecapture substrate, wherein the removeable capture substrate is in fluidcommunication with the secreted antibody and wherein the removeablecapture substrate is operable to bind the secreted antibody; incubatingthe secreted antibody with the removeable capture substrate to produce abound antibody; and measuring a binding interaction between the secretedantibody and the antigen.
 47. The method of claim 46, wherein the singleAPC is a single primary plasma cell.
 48. The method of claim 46, whereinthe single APC is an activated B cell.
 49. The method of claim 46,wherein the antigen is a cell or a cell fragment.
 50. The method ofclaim 46, wherein the antigen is a virus or a bacterial cell.
 51. Themethod of claim 46, wherein the antigen is a bacterial antigen or aviral antigen.
 52. The method of claim 46, wherein the antigen isfluorescently labeled.
 53. The method of claim 46, wherein measuring thebinding interaction comprises measuring a binding kinetic propertybetween the secreted antibody and the antigen.
 54. The method of claim53, wherein the binding kinetic property is a K_(on) rate; a K_(off)rate, a dissociation constant, or a combination thereof.
 55. The methodof claim 46, wherein measuring the binding interaction comprisesmeasuring the affinity of the secreted antibody and the antigen.
 56. Themethod of claim 46, wherein measuring the binding interaction comprisesmeasuring the avidity of the secreted antibody and the antigen.
 57. Themethod of claim 46, wherein the single ASC is maintained in the chamberwith the removeable capture substrate.
 58. The method of claim 46,wherein the removeable capture substrate is a microsphere.
 59. Themethod of claim 46, wherein the removeable capture substrate is ananosphere.
 60. The method of claim 46, wherein the removeable capturesubstrate is a polystyrene bead or a silica bead.
 61. The method ofclaim 46, wherein the removeable capture substrate is a carboxylic acid(COOH) functionalized bead.
 62. The method of claim 46, wherein theremoveable capture substrate is an anti-Ig bead.
 63. The method of claim46, wherein the removeable capture substrate is a Protein A coated bead.64. The method of claim 46, wherein the removeable capture substrate iscapable of binding the secreted antibody and the nucleic acids encodingthe secreted antibody.
 65. The method of claim 46, wherein measuring thebinding interaction comprises fluorescence imaging of the secretedantibody binding to the antigen.
 66. The method of claim 46, whereinmeasuring the binding interaction comprises surface plasmon resonance(SPR) spectroscopy, fluorescence anisotropy, interferometry orfluorescence resonance energy transfer (FRET).
 67. The method of claim46, further comprising performing a reverse transcription polymerasechain reaction (RT-PCR) on the nucleic acid encoding the secretedantibody to amplify the heavy and light chain genes of the antibody.